Methods for identifying substances with a potential herbicidal or growth-regulating action by means of plant transporter proteins, the use of the transporter proteins, and substances with a herbicidal and growth-regulating action

ABSTRACT

PCT No. PCT/EP94/04174 Sec. 371 Date Aug. 5, 1996 Sec. 102(e) Date Aug. 5, 1996 PCT Filed Dec. 15, 1994 PCT Pub. No. WO95/16913 PCT Pub. Date Jun. 22, 1995A method for identifying substances with a potential herbicidal and growth-regulating action by means of plant transporter proteins (transporters), the use thereof and substances identifiable by the method and having a herbicidal and growth-regulating action are described.

The present invention relates to methods for identifying substances witha potential herbicidal or growth-regulating action by means of planttransporter proteins (transporters), to the use thereof, and tosubstances identifiable by the method and having a herbicidal orgrowth-regulating action.

Novel crop protection agents are required to have lower toxicity,greater environmental compatibility and improved efficacy by comparisonwith conventional agents. is The probability of successfully findingsuch substances depends, besides the number of substances available,essentially on the method used to identify the active substances. Sinceconventional methods are usually based on direct testing of substancesfor their herbicidal or growth-regulating activity on plants, thesemethods are, a rule, very time-consuming and costly and requireextensive trial areas. Consequently, comparatively few substances can betested at the same time.

There is therefore a need for methods which can be carried out simplyand rapidly in order to permit a sufficient number of substances to beinvestigated.

We have now found a method which can be used to identify chemicalcompounds which specifically interact with a transporter protein fromplants. The method comprises, according to the invention, initiallytesting these substances on a transgenic organism, preferably aunicellular organism, which functionally expresses a plant transporterprotein, or on transgenic cells which functionally express a planttransporter protein, for inhibition of the transport process.

The substances identified thereby as inhibitory are subsequentlyinvestigated for their effect on whole plants. The term transporterproteins means for the purposes of this invention proteins which areresponsible for transporting substances through membranes in plantcells. In the method according to the invention, a plant transporterprotein is integrated into a test system which makes it possible todetermine, qualitatively and quantitatively, with biochemical,microbiological and physiological methods of measurement, the membranetransport function. The use of this test system makes it possiblespecifically to find active substances which are able to interact withthe transport proteins of the plant. Interaction of substances with atransporter protein may bring about inhibition or inactivation of thetransport process and, moreover, may lead to transport of the substanceitself. Since transport processes play a central part in overall plantmetabolism and are often essential for growth of plants, the methodaccording to the invention makes it possible to identify, specificallyand with higher probability, substances which exert an effect on plantgrowth. The interaction may elicit a growth-regulating or herbicidalaction if the substance which is found leads to inhibition of thenatural transport process. In the event that the substance withinhibitory activity is itself transported, it may, as ingredient of cropprotection agents, such as fungicides, insecticides, nematicides andacaricides, especially herbicides and growth regulators, increase theirmobility in plants and thus lead to novel agents which are moreeffective. The test system according to the invention can furthermore beused to investigate plant transporter proteins at the molecular level.

To date no methods have been described for identifying substances with aherbicidal or growth-regulating action by means of a plant transporterprotein. There has likewise been no description of the inhibition ofplant transport processes as mechanism of action of herbicides.Furthermore, it is also unknown whether plant transporter proteinsrepresent, because of their position in the metabolism, eg. in supplyingthe organs of reproduction, an interesting potential target forherbicides or growth regulators.

The method according to the invention comprises a biochemical testsystem which, in its first stage, is preferably carried out onunicellular organisms or on cells maintained in cell culture. This testsystem has the advantage that, by comparison with conventional methodsfor identifying herbicidal or growth-regulating substances, it can becarried out rapidly and straight-forwardly. It further makes it possibleto investigate, with little expenditure of time, a large number ofsubstances for their herbicidal or growth-regulating action. The methodaccording to the invention furthermore provides the advantage that itcan be used specifically to identify those substances which interactwith a very particular plant protein. In order to avoid unwanted effectson humans, animals and the environment, the target protein in the methodcan be chosen so that its function is specific for plants.

The proteins preferably employed as target proteins in the methodaccording to the invention are those responsible in plants fortransporting substances through membranes (transporter proteins),preferably those transporter proteins which are specific for plants.

The present invention thus relates to a method for identifyingsubstances which have a potential herbicidal or growth-regulating actionwhich arises due to inhibition or inactivation of a plant transportprocess, which comprises testing a chemical compound on a planttransporter protein for inhibition of the transport process, and thentesting the compound which is active therein for its herbicidal orgrowth-regulating activity on plants, or comprises

a) initially preparing the transporter protein by heterologousexpression of a DNA sequence which codes for this transport protein in atransgenic organism or transgenic cells, subsequently

b) employing this recombinant organism in its entirety or the transgeniccells for investigating a chemical compound for its inhibitory effect onsaid transporter protein, and

c) additionally investigating the compound for activity on the organismor cell which does not produce the corresponding transporter, in orderto preclude the possibility that the compound also has an inhibitoryeffect on other mechanisms in this organism or in the cell, and finally

d) testing the compound which is active against the transporter for itsherbicidal or growth-regulating activity on plants.

It is possible in general to use in the method according to theinvention all transporter proteins which occur in plants, and the DNAsequences which code for the transporter proteins.

Various transporter proteins responsible for transporting substancesthrough membranes have already been identified in plants, and in somecases DNA sequences which code for such transporter proteins areavailable.

Thus, for example, it has been possible to detect sucrose transportersdirectly on intact plants or on isolated leaf tissue. The sucrose uptakeis in this case pH-dependent (Giaquinta, Nature 267: 369-370, 1977,Annu. Rev. Plant Physiol. 34: 347-387, 1983; Delrot & Bonnemain, PlantPhysiol. 67: 560-564, 1981; Delrot, Plant Physiol. 67: 560-564, 1981).p-Chloromercuribenzyl-sulfonic acid and diethyl pyrocarbonate are highlyeffective transport inhibitors in this connection (Bush, 1989, PlantPhysiol. 89: 1318-1323). cDNA sequences which code for plant sucrosetransporters have already been described, for example for potatoes (p 62and StSUT1) and spinach (S21 and SoSUT1) (WO 94/00574; Riesmeier et al.,1993, Plant Cell 5:1591-1598; Riesmeier et al., 1992, EMBO J. 11:4705-4713), for Arabidopsis thaliana (suc1 and suc2 genes; EMBL genebank: Access No. X75365), Plantago major (EMBL gene bank: Access No.X75764), L. esculentum (EMBL gene bank: Access No. X82275) and Nicotianatabacum (EMBL gene bank: Access Nos. X82276 and X82277). In the case ofthe sucrose transporters, it was possible to clone cDNA sequences codingfor these transporters from spinach and potato by developing anartificial complementation system in Saccharomyces cerevisiae (Riesmeieret al., EMBO J. 11: 4705-4713, 1992; Riesmeier et al., 1993, Plant Cell5: 1591-1598).

Amino-acid transporters have likewise been identified in plants. Thegreen alga Chlorella which is related to higher plants, has at leastthree different regulated amino-acid transport systems (Sauer andTanner, Plant Physiol. 79: 760-764, 1985). This comprises activetransport provided with energy from a proton gradient generated byH⊕-ATPase. In higher plants it has been concluded indirectly frominvestigation of the composition of xylem and phloem that a passivetransport (facilitated diffusion) exists (Riens et al., Plant Physiol.97: 227-233, 1991). In contrast to this, the phloem or xylem of ricinuscotyledons or roots, respectively, is loaded with amino acidsselectively and counter to a concentration gradient (Schobert and Komor,Planta 177: 342-349, 1989; Planta 181: 85-90, 1990). It has beenpossible to demonstrate the existence of at least four independent H⊕cotransporters in isolated vesicles in various plant species (Li andBush, Plant Physiol. 94: 268-277, 1991). It was possible bycomple-mentation of an amino-acid transport mutant of yeast to isolateand characterize ureide and amino-acid permease genes from Arabidopsisthaliana, for example cDNA sequences which code for the amino-acidtransporters AAP1 and AAP2 (Frommer et al., 1993, Proc. Natl. Acad. Sci.USA 90:5944-5948; Kwart et al., 1993, Planta J. 4:993-1002; WO94/01559). It was possible, with the aid of a complementation methodusing the yeast mutant shr3, which is no longer able to directendogenous amino-acid transporters to the cell membrane (Ljungdahl etal., 1992, Cell 71: 463-478), to isolate a number of other DNA sequenceswhich code for amino-acid transporters from plants, eg. cDNA sequenceswhich code for the amino-acid transporters AAP3 (EMBL gene bank, AccessNumber: X77499), AAP4 (EMBL gene bank, Access Number: X77500), AAP5(EMBL gene bank, Access Number: X77501), AAT1 (EMBL gene bank, AccessNumber: X71787) and NTR1 (EMBL gene bank, Access Number: X77503) fromArabidopsis thaliana. Also known are cDNA sequences which code for plantammonium transporters, for example a cDNA coding for the ammoniumtransporter AMT1 from Arabidopsis thaliana (Ninnemann et al., 1994, EMBOJ. 13:3464-3471; German Patent Application P 43 37 597.9; EMBL genebank, Access Number: X75879).

The cloning of genes for membrane-bound transporter proteins has alreadybeen described several times, besides the abovementioned examples. It ispossible in principle for several different routes to be followed forcloning the genes of membrane proteins. For example, in the isolation ofthe glucose transporter gene from erythrocytes, it was possible toidentify cDNA clones after purification of the protein (Mueckler et al.,Science 229: 941-945, 1985). However, in many cases, it is so difficultto purify membrane transporters that other methods have to be used, eg.heterologous expression in oocytes (Hediger et al., Nature 330: 379-381,1987). Plant plasmalemma H⊕-ATPase genes have been cloned via homologywith animal and fungal genes. It has been possible to isolate plantglucose transporter genes from Chlorella by differential cDNA screening(Sauer et al., EMBO J. 8: 3045-3050, 1990). A cDNA clone from Chlorellahas been used as heterologous probe for the cloning of several glucosetransporter genes from higher plants (Sauer et al., 1990). Thechloroplastidic triose phosphate translocator (TPT) was radiolabeled viathe inhibitor DIDS, and the labeled protein was purified and partiallysequenced. Synthetic oligonucleotides derived from the partial peptidesequences were used as probes for isolating TPT-encoding cDNAs (Fluggeet al., EMBO J. 8: 39-46, 1989). The DNA sequences already known to codefor plant transporter proteins can in turn be used to identify andisolate other DNA sequences which code for transporter proteins fromplants by means of conventional techniques of molecular biology.

Genes preferably used in the method according to the invention code fortransporter proteins which are essential for the growth of the plants.Inhibition of the corresponding transporter protein should accordinglylead to impairment of growth. On the assumption that DNA sequences whichcode for the corresponding transporter protein are available, thisdetection is possible, for example, by means of expression or ofantisense inhibition of the corresponding gene in transgenic plants(Willmitzer, Trends Genet. 4: 13-18, 1988). The techniques for producingsuch transgenic plants are known to a skilled person. Thus, for example,it has been possible to show, by expression of an antisense RNA whichcodes for the triose phosphate translocator, that even a small reductionin the expression of the protein leads to a drastic inhibition of growthof the plant (Riesmeier et al., Proc. Natl. Acad. Sci. USA 90:6160-6164, 1993). It has likewise been possible to show for the sucrosetransporter that a reduction in the activity leads to a great inhibitionof growth of potato plants. Furthermore, the leaves of the affectedplants are damaged, and the plants produce few or no potato tubers(Riesmeier et al., 1994, EMBO J. 13: 1-7). Since the formation or theorgans of reproduction is greatly impaired, it can be expected that asuitable herbicide not only inhibits the growth of a plant but alsoimpedes its reproduction. The same is to be expected in the case ofammonium and amino-acid transport because these have an essentialfunction in metabolism for the transport of nitrogen.

On the basis of the described essential function, preferred embodimentsof the present invention provide for the use of the transporter proteinsfor amino acids, sucrose and ammonium and as target proteins for findingpotential active substances specific for plants, especially the use ofthe transporter proteins specified above.

Suitable genes coding for transporter proteins are introduced, with theaid of conventional methods of molecular genetics, into an organism orinto cells in such a way that expression of a transporter proteincapable of functioning is ensured.

The organism mentioned in step a) in the method is preferably aunicellular organism. The unicellular organism is chosen so that itscells can easily be cultivated and are suitable for expressing thetransporter protein. Particularly suitable for this purpose aremicroorganisms such as bacteria, fungi or yeasts. However, it is alsopossible to use single cells of an organism. Also suitable for use inthe method according to the invention are, for example, plant cellsmaintained in cell culture, or callus cultures, as well as animal cellsin cell culture, especially including oocytes, preferably Xenopusoocytes. The transgenic cells obtained by introducing a planttransporter protein gene, or the recombinant organism obtained, can thenas a whole be part of a test system or can be used to isolate membranesor purified transporter protein. The invention likewise relates to theserecombinant organisms (bacteria, fungi and yeasts) and transgenic cells.A gene which codes for a transporter protein can additionally beintroduced into a particular mutant of an organism which is unable togrow without the functioning of the corresponding transporter protein(Riesmeier et al., EMBO J. 11: 4705-4713, 1992). The use of such amutant has the particular advantage that it makes it possible for growthof the recombinant organism in a suitable medium to serve as a measureof the functioning of the transporter, and thus to describequantitatively the effect of the substances to be investigated onmembrane transport. The growth test is distinguished by beingparticularly simple to operate and by rapid throughput of substances. Itis therefore preferred to use suitable mutants as have been described,for example, for sucrose transporters (SUSY7; see Riesmeier et al., EMBOJ. 11: 4705-4713, 1992), aminoacid transporters (yeast mutants 22574dand JT16; see Frommer et al., Proc. Natl. Acad. Sci. USA 90: 5944-5948,1993; yeast mutant shr3; see Ljungdahl et al., 1992, Cell 71: 463-478)and ammonium transporters (Ninnemann et al., 1994, EMBO J. 13:3464-3471).

The appropriate transporter gene is used according to the invention totransform said organism or cells.

The recombinant organism or the transgenic cells can then be multipliedas desired by conventional microbiological methods and are thusavailable to an unrestricted extent for use in the test system. Amultistage procedure is preferred for testing substances for herbicidaland/or growth-regulating properties: firstly recombinant organisms ortransgenic cells are cultured in a medium in which an essential growthsubstrate is chosen so that it enters the cells only through thetransporter to be investigated and, in addition, cannot be functionallyreplaced by any other substrate in the medium. In the case of a sucrosetransporter, for example, the medium contains sucrose as the sole carbonsource, in the case of an amino-acid transporter the medium contains,for example, an amino acid which serves as sole carbon or nitrogensource for the cells, and in the case of an ammonium transporter themedium contains ammonium as sole nitrogen source. The substances to beinvestigated are added to the medium during the growth phase, and thegrowth of the cells is determined by conventional methods.

In order to detect a specific interaction of a substance with a membranetransporter, and in order to preclude other modes of action as cause ofthe inhibition of growth, the substance must meet the followingconditions:

1) Recombinant cells must show distinctly less growth after addition ofthe substance than without addition of the substance.

2) The same organisms must not be inhibited in growth after addition ofthe substance if another growth substrate is present in the medium, isable functionally to replace the actual substrate of the transporter inthe cells, and itself does not enter the cells via the correspondingtransporter.

Yeast cells are able, for example, to grow either with sucrose or withan amino acid as carbon source. Yeasts are able to utilize either anamino acid or ammonium ions as nitrogen source.

If a substance meets the stated conditions, it can be furtherinvestigated in a biochemical test. This test measures the membranepassage of the natural substrate or of the inhibiting substance. It ispossible to employ for this purpose whole yeast cells or isolatedmembrane vesicles. Membrane passage of the transporter substrate or ofthe inhibiting substance can be detected by the substrate or thesubstance being isolated from the cells or membrane vesicles which havebeen separated off, and being detected by conventional analyticalmethods. As a rule, the substrate or the substance will be added inradiolabeled form and subsequently analyzed via its radioactiveemission. The type of inhibition can be determined by altering theconcentrations of the substrate and of the inhibiting substance usingconventional biochemical methods. It is furthermore possible to detectthereby whether the substance itself is transported by the transporter.

If a substance meets the abovementioned conditions, it can beinvestigated, with or without further biochemical investigations,directly on whole plants or suitable parts of plants for its herbicidalaction or for its mobility in the plant. Conventional herbological andphysiological methods can be used for this purpose.

The invention likewise relates to the substances which can be identifiedby the method according to the invention and which have a herbicidaland/or growth-regulating action on plants, and to the formulationthereof with other herbicides, growth regulators, nematicides,insecticides, acaricides, fungicides and auxiliaries conventional inagriculture.

The method according to the invention can also be used to identifysubstances which are themselves conveyed by the transporter protein(transporter) through the plant cell membrane. The invention likewiserelates to these substances. The method can thus be used to identifythose chemical structures which are transported particularly well in theplant. This property of good mobility is particular desired for cropprotection agents such as herbicides and insecticides.

The method according to the invention can also be used as test systemfor identifying transportable chemical structures. Since sucrose andamino-acid transporters represent the main transport systems for organicmolecules in the membranes, it is possible, for example, to utilize theyeast system as simple test for investigating the mobility of organicsubstances in the plant. The preferred procedure in this connection ismultistage: it is possible in the first place to investigate the uptakeof foreign substances in a growth test. It is then possible, building onthis, to employ direct measurements of transport in intact yeasts orisolated membranes for more detailed investigation. Yeasts which do notcontain the transporter are always available as control. Besides yeasts,it is also possible to use for this purpose other organisms, especiallyunicellular organisms, and especially cell cultures of animal or plantcells.

In the event of specific impairment of transport there should beobserved to be a reduction in growth of the transgenic yeasts. It ispossible by investigating the transporters to gain a betterunderstanding of which properties of a substance are necessary fortransport-ability. It would be possible for a number of substances whichhave hitherto been ineffective as pesticides because of a lack oftransportability to alter the molecules chemically so that they acquirethese properties of a substance and thus reach their site of actionbetter. It would likewise be conceivable to alter the transporters bymethods of molecular biology or by mutation in such a way that they arebetter able to transport the pesticides, such as insecticides andherbicides, through membranes. The altered transporter protein genes canthen in turn be introduced into plants.

The present invention likewise relates to the use of such transporterproteins which transport pesticides through plant cell membranes.

The invention likewise relates to pesticides, especially herbicides andgrowth regulators, which are able to inhibit the plant transport system,where the plant transport system (transporter) is, for example, thesucrose, the ammonium or the amino-acid transporter.

Besides the finding of substances, the system can also be employed forinvestigating other important questions, eg. what are the structures ofsuch proteins and how the substrates are recognized, or sic! and inwhich connection the structure is related to the function, and howgenerally substances are distributed in an organism. The method whichhas led to isolation and characterization of the described transporterscan, however, also be employed for isolation of other proteins (eg. iontransporters or transport proteins from animal systems) and forinvestigation in the same manner.

The following examples of use illustrate the subject-matter of theinvention without restricting it. The use of plant sucrose, amino-acidand ammonium transporters for identifying substances which haveinhibitory effects on these transporters is described.

EXAMPLES OF USE Example 1

Identification of inhibitors of slant amino-acid transporters

Transporter investigations are carried out on the yeast mutant 22574d(Jauniaux & Grenson, Eur. J. Biochem. 190: 39-44, 1990), which harbors amutation in the gene for amino-acid permease, and on the yeast mutant JT16 (Tanaka and Fink, Gene 38: 205-214, 1985) which is incapable ofhistidine uptake, for identification of inhibitors of plant amino-acidtransporters. These mutants are transformed with cDNA sequences whichcode for plant amino-acid transporters and lead to expression offunctional transporters in the yeast cells. The cDNA sequences are cDNAsequences which code for the amino-acid transporters AAP1 and AAP2 fromArabidopsis thaliana (Frommer et al., 1993, Proc. Natl. Acad. Sci. USA90: 5944-5948; Kwart et al., 1993, Plant J. 4:993-1002). For themeasurements, cells of these mutants are cultured in minimal medium(NAAG+5 mmol/l proline) at 28° C. and harvested in the logarithmic phase(OD₆₀₀ =0.6). The cells are spun down in a centrifuge at 4000 rpm and 4degrees Celsius for 10 minutes and washed 2× with AUB buffer (modifiedAAB buffer, Ljungdahl et al.). The cell concentration is adjusted to anOD₆₀₀ of 25 in AUB buffer.

    ______________________________________                                        Media and buffer used:                                                        ______________________________________                                        NAAG medium: 1.7 g/l yeast nitrogen base,                                                  w/o amino acids                                                  and          ammonium sulfate                                                 SDG1u medium:                                                                              10 g/l glucose                                                                20 g/l agarose                                                                6.7 g/ lacuna! yeast nitrogen base w/o                                        amino acids                                                      SDsuc medium:                                                                              20 g/l gucose  sic!                                                           20 g/l agarose                                                                6.7 g/ lacuna! yeast nitrogen base w/o                                        amino acids                                                                   20 g/l sucrose                                                                20 g/l agarose                                                   AUB buffer:  10 mmol/l MES                                                                 2 mmol/l magnesium chloride                                                   0.6 mmol/l sorbitol                                              ______________________________________                                    

100 μl portions of the cell suspension are mixed with 100 μl of a 1mmol/l L-proline solution (18.5 kBq of L- 14C!-proline) and variousconcentrations of the appropriate substance to be investigated. After20, 60, 120 and 180 seconds, a 50 μl aliquot is removed, diluted in 4 ml-of ice-cold AUB buffer and filtered through glass fever sic! filter. Inorder to remove nonspecifically bound L- 14C!-proline, the cells arewashed 2× with 4 ml of ice-cold water each time. The radioactivityretained on the glass filter is then measured in a liquid scintillationcounter. The radioactivity reflects the amount of radiolabeled prolinetaken up by the cells. Uptake of radiolabeled proline without additionof a potential inhibitor is determined as one control. Cells of each ofthe mutants 22574d and JY 16 which have not been transformed with cDNAsequences coding for plant amino-acid transporters are used as furthercontrol. Results of measurements of this type on the mutants 22457d-AAP2which expresses the amino-acid transporter AAP2 from Arabisopsis sic!thaliana are shown in the following table.

    ______________________________________                                        Tested                  Activity relative to                                  substance    Concentration                                                                            control  %!                                           ______________________________________                                        CCCP         10     μM   15.6 ± 2.1                                     DNP          0.1    mM      7.6 ± 1.6                                      DEPC         1      mM      3.1 ± 1.2                                      Azetidine-2- 10     mM      62                                                carboxylate                                                                   D-Proline    10     mM      90                                                ______________________________________                                    

For the investigations, yeast cells of the strain 22457d weretransformed with a cDNA sequence which codes for the amino-acidtransporter AAP2, and the uptake of radiolabeled proline in the presenceof various substances was determined. The various substances tested,their concentration, and the activity of the transporter, shown aspercentage activity of the control with which no potential inhibitorswere added, are indicated.

Substances which specifically show an inhibitory effect on theamino-acid transporter AAP2 are tested on whole plants for theirherbicidal and growth-regulating effect.

Example 2

Identification of inhibitors of the sucrose transporter from Spinaciaoleracea

Yeast cells of the strain SUSY7 (Riesmeier et al., 1992, EMBO J. 11:4705-4713) are used to identify substances which have an inhibitoryeffect on sucrose transporters from spinach. Cells of this strain aretransformed with a cDNA sequence which codes for the sucrose transporterS21 (SoSUT1) from spinach and which ensures expression of a functionaltransporter in the cells. The yeast cells are cultured in minimal medium(SD+2% (w/v) sucrose, pH 3.8) at 28° C. and harvested in the logarithmicphase (OD₆₀₀ =0.6). The cells are spun down at 4000 rpm for 10 minutesand washed twice with SD medium. A cell concentration of c=50 g/l in SDmedium is adjusted according to the wet weight of the cells, and 200 μlaliquot fractions are taken. Before the actual reaction, the cells areincubated in 10 mM glucose for 5 min and adjusted to pH 3.8 with MESbuffer. The reaction is started by adding 200 μl of a 0.2 mmol/l14C!-sucrose solution (3 mCi/mmol) in SD medium (pH 3.8). After 20, 60,120, 180 and 240 seconds, 70 μl portions of the suspension are removedand pipetted into 4 ml of ice-cold water in order to stop the reaction.The cells are filtered off on glass fiber filters and washed twice withice-cold water. The radioactivity on the filters is subsequentlydetermined in a liquid scintillation counter. The substances to betested are added 30 seconds before addition of the sucrose. The uptakeof radiolabeled sucrose without addition of a potential inhibitor isdetermined as one control. Cells of the strain SUSY7 which are nottransformed with cDNA sequences coding for a sucrose transporter fromspinach are employed as further control. The results of transportmeasurements of this type are shown in the following table.

    ______________________________________                                                                    Activity relative                                 Tested                      to the control                                    substances      Concentration                                                                              %!                                               ______________________________________                                        CCCP            10      μM   10                                            PCMBS           0.1     mM      21                                            DEPC            0.5     mM      6                                             Palatinose      2       mM      85.0                                          Mannose         2       mM      86.5                                          Tagatose        2       mM      104.7                                         Melizitose  sic!                                                                              2       mM      97.2                                          Raffinose       2       mM      110.3                                         Galactose       2       mM      97.7                                          Cellobiose      2       mM      99.4                                          Melibiose       2       mM      92.3                                          Altrose         2       mM      80.6                                          α-Lactose 2       mM      81.8                                          β-Lactose  2       mM      94.9                                          α-Phenylglucose                                                                         2       mM      8.0                                            ##STR1##       1.0     mmol/l  0                                              ##STR2##       1.0     mmol/l  0                                             ______________________________________                                    

The various tested substances, their concentration and the activity ofthe transporter, shown as percentage activity of the control in which nopotential inhibitors were added, are indicated. Substances whichspecifically show an inhibitory effect on the sucrose transporter S21(SoSUT1), are tested for their herbicidal and growth-regulating effecton whole plants. Some of the identified inhibitors of the sucrosetransporter have herbicidal activity on plants, for example PCMBS.

Example 3

Identification of inhibitors of the sucrose transporter from Solanumtuberosum

Yeast cells of the strain SUSY7 (Riesmeier et al., 1992, EMBO J. 11:4705-4713) are used to identify substances which have an inhibitoryeffect on sucrose transporters from potato. Cells of this strain aretransformed with a cDNA sequence which codes for the sucrose transporterP62 (StSUT1) from potato (WO 94/00574, Riesmeier et al., 1993, PlantCell 5:1591-1598) and which ensures expression of a functionaltransporter in the cells. The resulting yeast strain was calledSUSY-7-P62 (StSUT1). The yeast cells are cultured in minimal medium (SD+2% (w/v) sucrose, pH 3.8) at 28° C. and harvested in the logarithmicphase (OD₆₀₀ =0.6). The cells are spun down at 4000 rpm for 10 minutesand washed twice with SD medium. A cell concentration of c=50 g/l in SDmedium is adjusted according to the wet weight of the cells, and 200 Alaliquot fractions are taken. Before the actual reaction, the cells areincubated in 10 mM glucose for 5 min and adjusted to pH 3.8 with MESbuffer. The reaction is started by adding 200 Al of a 0.2 mmol/l14C!-sucrose solution (3 mCi/mmol) in SD medium (pH 3.8). After 20, 60,120, 180 and 240 seconds, 70 μl portions of the suspension are removedand pipetted into 4 ml of ice-cold water in order to stop the reaction.The cells are filtered off on glass fiber filters and washed twice withice-cold water. The radioactivity on the filters is subsequentlydetermined in a liquid scintillation counter. The substances to betested are added 30 seconds before addition of the sucrose. The uptakeof radiolabeled sucrose without addition of a potential inhibitor isdetermined as one control. Cells of the strain SUSY7 which are nottransformed with cDNA sequences coding for a sucrose transporter frompotato are employed as further control. The results of transportmeasurements of this type are shown in the following table.

    ______________________________________                                        Tested                    Activity relative to                                substances     Concentration                                                                            the control  %!                                     ______________________________________                                        CCCP           10     μM   9                                               PCMBS          0.1    mM      20                                              2,4-DNP        0.1    mM      3                                               DEPC           0.5    mM      6                                               N-Ethylmaleimide                                                                             1      mM      22                                              Palatinose     2      mM      102                                             Tagatosa       2      mM      103                                             Raffinose      2      mM      110                                             β-Lactose 2      mM      91                                              α-Phenylglucose                                                                        2      mM      7                                               ______________________________________                                    

The various tested substances, their concentration and the activity ofthe transporter, shown as percentage activity of the control in which nopotential inhibitors were added, are indicated.

Substances which specifically show an inhibitory effect on the sucrosetransporter P62 (StSUT1), are tested for their herbicidal andgrowth-regulating effect on whole plants. Some of the identifiedinhibitors of the sucrose transporter have herbicidal activity onplants, for example PCMBS.

Example 4

Determination of the substrate specificity of the sucrose transporter

The investigations of the substrate specificity and the K_(m)determination are carried out in the yeast strain SUSY7-pP62 (StSUT1)(Riesmeier et al., 1993, Plant Cell 5:1591-1598), which expresses thesucrose transporter from Solanum tuberosum. This entails carrying outthe method of Example 3 apart from the fact that the substrateconcentration is changed and no potential inhibitors are added. Tocontrol for the background activity, the uptake of 14C!-sucrose by theyeast strain SUSY7 which does not express the transporter is determined.This strain shows no measurable uptake of 14C!-sucrose over themeasurement period. Test results:

    ______________________________________                                               K.sub.m for sucrose                                                                      1 mM                                                               K.sub.m for maltose                                                                     10 mM                                                        ______________________________________                                    

Example 5

Activation of sucrose transport

The transport of sucrose by the sucrose transporter P62 (StSUT1) fromSolanum tuberosum can be increased by previous energizing of the yeastcells by incubation in glucose, stachyose and adenine. The same increasein transport activity is achieved by reducing the pH to pH 3.8 in themeasurement. The measurement of transport to determine the activationtakes place as described in Example 3 but the cells are incubated beforethe actual measurement not in 10 mM glucose for 5 min but in solutionsof various concentrations of glucose, stachyose and adenine for 5 min.Yeast cells which are not incubated in solutions of these substancesbefore the measurement act as control. The following table indicates theextent of activation of sucrose transport with various concentrations ofglucose, stachyose and adenine compared with the control.

    ______________________________________                                        Activity relative to the control  %!                                                      c1  0.2 mol/l!                                                                         c2  2 mmol/l!                                            ______________________________________                                        Glucose        89 ± 5  90 ± 3                                           Stachyose     144 ± 9 101 ± 8                                           Adenine       141 ± 5 225 ± 6                                           ______________________________________                                    

Stachyose stimulates sucrose transport at low concentrations, while thiseffect is reversed at high concentrations (c>0.4 mmol/l), and stachyosebegins to reduce the uptake. For adenine there is a linear relationbetween the adenine concentration and the increase in the sucrosetransport rate. It can be shown by competition studies which domains areimportant for the affinity of the transporter for sucrose.

Example 6

Identification of inhibitors of Plant ammonium transporters

The measurements of transport identify inhibitors of plant ammoniumtransporters are carried out with the structural analog methylaminebecause radiolabeled ammonium is not commercially obtainable.

Yeasts of the yeast strain ♮ 26972c (Dubois & Grenson, Mol. Gen. Genet.175: 67-76, 1979) which harbor mutations in the NH₄ ⁺ permease genesMEP1 and MEP2 are used for the investigations.

The yeast cells are transformed by standard methods with cDNA sequenceswhich code for an ammonium transporter from plants and which permitexpression of a functional transporter in the yeast cells. This is thetransporter AMT1 from Arabidopsis thaliana (Ninnemann et al., 1994, EMBOJ. 13:3464-3471). The cells are cultured in NAAG-medium (2% glucose, 1.7g/l yeast nitrogen-base w/o amino acids and ammonium sulfate (Difco),supplemented with 500 μg/ml L-proline) at 28 degrees Celsius, andharvested in the logarithmic phase (OD₆₆₀.=0.6). The cells are then spundown in a centrifuge at 4000 rpm and 4 degrees Celsius for 10 minutes,washed twice with 20 mM sodium phosphate buffer, pH 7, and taken up inthe same buffer to an 0D₆₆₀ of 8. 200 μl aliquot fractions of the cellsuspension are taken. 5 minutes before the actual measurement, theyeasts are activated by addition of 100 mM glucose and incubated at 30degrees Celsius. To start the reaction, 100 μl of the cell suspensionare added to 100 l of reaction mixture (20 mM Na phosphate buffer, pH7;18.5 kBq of ¹⁴ C!-methylamine (NEN); 100 M methylamine; substance to betested depending on the experiment (see FIG. 4)). In each case 10, 60,120 and 180 seconds after starting the reaction, 50 μl aliquots aretaken, added to 4 ml of ice-cold 5 mM methylamine solution and filteredon glass fibre filters. After the filters have been washed with afurther 8 ml of a methylamine solution, the radioactivity on the filtersis subsequently determined in a liquid scintillation counter. Inhibitorsare added 60 seconds before adding the glucose.

The uptake of radiolabeled methylamine without addition of a potentialinhibitor is determined as one control. The same transportinvestigations are carried out on yeast cells of the strain Σ26972cwhich have not been transformed as further control.

The results of investigations of this type are shown in the followingtable.

    ______________________________________                                        Tested                   Activity relative                                    substances    Concentration                                                                            to the control  %!                                   ______________________________________                                        None          /          100                                                  Methylamine   500    μM   40                                               Dimethylamine 500    μM   89                                               Trimethylamine                                                                              500    μM   89                                               Ethylamine    500    μM   93                                               KCl           500    μM   98                                               RbCl          500    μM   96                                               CsCl          500    μM   98                                               NH.sub.4 Cl   500    μM   10                                               Cycloheximide 10     μg/ml                                                                              85                                               Antimycin A   10     μg/ml                                                                              8                                                DCCD          200    μM   15                                               2-4 DNP  sic! 100    μM   18                                               CCCP          10     μM   40                                               ______________________________________                                    

The various tested substances, their concentration and the activity ofthe transporter, shown as percentage activity of the control in which nopotential inhibitors were added, are indicated.

Substances which specifically show an inhibitory effect on the ammoniumtransporter AMT1, are tested for their herbicidal and group-regulatingeffect on whole plants.

Some of the identified inhibitors of the ammonium transporter haveherbicidal activity on plants, for example methylamine.

Example 7

Determination of the substrate specificity of the ammonium transporterAMT1 from Arabidopsis thaliana

The investigations of the substrate specificity and the K_(m)determinations are carried out with the yeast strain ♮ 26972c (Dubois &Grenson, Mol. Gen. Genet. 175: 67-76, 1979) which expresses the plantammonium transporter AMT1. The method for this is as in Example 6,except for the fact that the methylamine substrate concentration isvaried. To control for the background activity, the uptake of ¹⁴C!-methylamine by a yeast strain which does not express said transporteris determined. To determine the affinity of the transport system forammonium, the inhibition of methylamine transport by variousconcentration sic! of ammonium is determined (inhibitor constant, K_(i))

    ______________________________________                                        K.sub.m for methylamine                                                                              65 μM                                               K.sub.i for ammonium   <10 μM                                              ______________________________________                                    

We claim:
 1. A method for identifying substances which have a potentialherbicidal or growth-regulating action which arises due to inhibition orinactivation of a plant transport process, which comprises.a) initiallypreparing a transporter protein by heterologous expression of a DNAsequence which codes for said transporter protein in a transgenic plantfungus, yeast or eukaryotic cell, subsequently b) employing saidtransgenic plant, fungus, yeast or eukaryotic cell for assaying achemical compound for its inhibitory effect on said transporter protein,and c) additionally assaying the chemical compound for activity onplant, fungus, yeast or eukaryotic cell which do not produce thecorresponding transporter protein, in order to preclude the possibilitythat the chemical compound also has an inhibitory effect on othermechanisms in said plant, fungus, yeast or eukaryotic cell, and finallyd) testing the chemical compound which is active against the transporterprotein for its herbicidal or growth-regulating activity on plants.
 2. Amethod as claimed in claim 1, wherein the transporter is a sucrosetransporter.
 3. A method as claimed in claim 1, wherein the transporteris an amino-acid transporter.
 4. A method as claimed in claim 1, whereinthe transporter is an ammonium transporter.
 5. A method as claimed inclaim 1, wherein the organism is a fungus or yeast.
 6. A method asclaimed in claim 1, wherein the cells are plant cells.
 7. A method asclaimed in claim 1, wherein the cells are animal cells.
 8. A method asclaimed in claim 1, wherein the cells are oocytes.
 9. A method asclaimed in claim 1, wherein the cells are Xenopus oocytes.